HIV infection remains incurable owing to the persistence of viral reservoirs that are resistant to current antiviral therapies. The latent reseroir is rapidly established during acute/early infection, and during this phase many viral RNA+ cells lack activation and proliferation markers and thus resemble resting CD4+ T cells. These findings suggest that latency is often established by direct infection of resting T cells. In vitro models o HIV-1 latency frequently employ direct infection of resting CD4 T cells, including our recently described latency system utilizing IL-4 to render T cells infectible without inducing T cell activation. We have reported that when HIV-1 infects resting CD4 T cells, large numbers of latently infected cells are generated which contain HIV-1 genomes that have not integrated and cannot integrate, yet these genomes are capable of de novo virus production when the cell is activated several days after infection. Using an IL-4-based latency system, we find that cells containing only unintegrated HIV-1 constitute about one half of all latently infected cells. We fin that latent unintegrated genomes respond to shock and kill agents but that their responses indicate epigenetic regulation that differs from integrated proviruses. We hypothesize that this new form of latency is a common occurrence in standard in vitro models of HIV-1 latency. The studies we propose will define the mechanisms controlling this new form of latency and its contribution to in vitro models of HIV-1 latency. We further find that the Vpr gene of HIV-1 is a key regulator the chromatization of HIV-1 genomes, and we will define the mechanisms by which it performs this function. We find that Vpr is essential for expression from uDNA in resting T cells, and that without Vpr, these genomes are unresponsive to stimulation and display altered chromatin methylation. We hypothesize that Vpr counteracts cellular defenses against invading parasitic DNA that install transcriptionally repressive chromatin modifications. This project will provide new insights into the establishment and regulation of HIV-1 latency. This project will also provide necessary information on the constitution and functioning of important in vitro models of latency that must be accounted for in the development of novel anti-viral therapies.